Tuberculosis is a chronic infectious disease caused by infection with M. tuberculosis. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as an acute inflammation of the lungs, resulting in fever and a nonproductive cough. If untreated, serious complications and death typically result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behavior is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistance.
In order to control the spread of tuberculosis, effective vaccination, and accurate early diagnosis of the disease are of utmost importance. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common Mycobacterium employed for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of M. bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public with this agent.
Diagnosis of tuberculosis is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
While macrophages have been shown to act as the principal effectors of M. tuberculosis immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of M. tuberculosis in Acquired Immunodeficiency Syndrome patients, due to the depletion of CD4+ T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4+ T cells have been shown to be potent producers of gamma-interferon (IFN-xcex3), which, in turn, has been shown to trigger the anti-mycobacterial effects of macrophages in mice. While the role of IFN-xcex3 in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-xcex3 or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN-xcex3 stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, interleukin-12 (IL-12) has been shown to play a role in stimulating resistance to M. tuberculosis infection. For a review of the immunology of M. tuberculosis infection, see Chan and Kaufmann, 1994, Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, DC.
Accordingly, there is a need for improved vaccines, and methods for preventing and treating tuberculosis.
The present invention relates to fusion proteins of M. tuberculosis antigens. In particular, it relates to fusion polypeptides that contain two or three M. tuberculosis antigens, polynucleotides encoding such polypeptides, methods of using the polypeptides and polynucleotides in the treatment and prevention of M. tuberculosis infection.
The present invention is based, in part, on Applicants"" discovery that two polynucleotides, each containing three M. tuberculosis coding sequences, produced recombinant fusion proteins that retained the immunogenicity and antigenicity of their individual components. The fusion proteins induced both T cell and B cell responses, as measured by T cell proliferation, cytokine production, and antibody production. Furthermore, a fusion protein was used as an immunogen with adjuvants in vivo to elicit both cell-mediated and humoral immunity to M. tuberculosis. Additionally, a fusion protein of two antigens was made by a fusion construct and used in a vaccine formulation with an adjuvant to afford long-term protection in animals against the development of tuberculosis. The fusion protein was a more effective immunogen than a mixture of the two proteins.
In a specific embodiment of the invention, the isolated or purified M. tuberculosis polypeptides of the invention may be formulated as pharmaceutical compositions for administration into a subject in the prevention and/or treatment of M. tuberculosis infection. The immunogenicity of the fusion protein may be enhanced by the inclusion of an adjuvant.
In another aspect of the invention, the isolated or purified polynucleotides are used to produce recombinant fusion polypeptide antigens in vitro. Alternatively, the polynucleotides may be administered directly into a subject as DNA vaccines to cause antigen expression in the subject, and the subsequent induction of an anti-M. tuberculosis immune response.